1. Field of the Invention
The present invention relates to genes which play a part in the structural and functional regulation of chromatin, and their use in therapy and diagnosis.
2. Related Art
Higher-order chromatin is essential for epigenetic gene control and for the functional organization of chromosomes. Differences in higher-order chromatin structure have been linked with distinct covalent modifications of histone tails which regulate transcriptional ‘on’ or ‘off’ states and influence chromosome condensation and segregation.
Histones constitute a highly conserved family of proteins (H3, H4, H2A, H2B, H1) which are the major components of eucaryotic chromatin structure. Histones compact genomic DNA into basic repeating structural units, the nucleosomes. In addition to their DNA packaging function, histones have been proven to be integral components of the molecular machinery that regulates gene expression.
Post-translational modifications of histone N-termini, particularly of H4 and H3, are well-documented and have functionally been characterized as changes in acetylation, phosphorylation and, most recently, methylation. In contrast to the large number of described histone acetyltransferases (HATs) and histone deacetylases (HDACs), genes encoding enzymatic activities that regulate phosphorylation or methylation of histone N-termini are only beginning to be identified. Moreover, the interdependence of the different histone tail modifications for the integration of transcriptional output or higher-order chromatin organization is currently not understood.
Overall, there is increasing evidence that the regulation of normal and aberrant cellular proliferation is not only affected on the transcriptional level, but that also a higher level of regulation is involved, i.e., the organization of chromatin structure through the modification of histone molecules. The determination of the proteins and the molecular mechanisms involved in histone modification will contribute to the understanding of the cellular proliferation program and will thus shed light on the mechanisms involved in aberrant proliferation occurring in tumor formation and progression.
The functional organization of eucaryotic chromosomes in centromeres, telomeres and eu- and heterochromatic regions is a crucial mechanism for ensuring exact replication and distribution of genetic information on each cell division. By contrast, tumor cells are frequently characterized by chromosomal rearrangements, translocations and aneuploidy (Solomon, et al., Science 254:1153–1160 (1991); Pardue, Cell 66:427–431 (1991)).
Although the mechanisms which lead to increased chromosome instability in tumor cells have not yet been clarified, a number of experimental systems, beginning with telomeric positional effects in yeast (Renauld, et al., Genes & Dev. 7: 1133–1145 (1993); Buck and Shore, Genes & Dev. 9:370–384 (1995); Allshire, et al., Cell 76:157–169 (1994)), via positional effect variegation (PEV) in Drosophila (Reuter and Spierer, BioEssays 14:605–612 (1992)), and up to the analysis of translocation fracture points in human leukaemias (Solomon, et al., Science 254:1153–1160 (1991); Cleary, et al., Cell 66:619–622 (1991)), have made it possible to identify chromosomal proteins which are involved in causing deregulated proliferation.
First, it was found that the overexpression of a shortened version of the SIR4-protein leads to a longer life in yeast (Kennedy, et al., Cell 80:485–496 (1995)). Since SIR proteins contribute to the formation of multimeric complexes at the stationary mating type loci and at the telomere, it could be that overexpressed SIR4 interferes with these heterochromatin-like complexes, finally resulting in uncontrolled proliferation. This assumption accords with the frequency of occurrence of a deregulated telomere length in most types of human cancer (Counter, et al., Embo. J. 11:1921–1928 (1992)).
Second, genetic analyses of PEV in Drosophila have identified a number of gene products which alter the structure of chromatin at heterochromatic positions and within the homeotic gene cluster (Reuter and Spierer, BioEssays 14:605–612 (1992)). Mutations of some of these genes, such as modulo (Garzino, et al., Embo J. 11:4471–4479 (1992)) and polyhomeotic (Smouse and Perrimon, Dev. Biol. 139:169–185 (1990)), can cause deregulated cell proliferation or cell death in Drosophila. 
Third, mammalian homologues of both activators, e.g., trithorax or trx-group, and also repressors, e.g., polycomb or Pc-group, of the chromatin structure of homeotic Drosophila selector genes have been described. Among these, human HRX/ALL-1 (trx-group) has been shown to be involved in leukaemogenesis induced by translocation (Tkachuk, et al., Cell 71:691–700 (1992); Gu, et al., Cell 71:701–708 (1992)), and it has been shown that the overexpression of murine bmi (Pc-group) leads to the formation of lymphomas (Haupt, et al., Cell 65:753–763 (1991); Brunk, et al., Nature 353:351–355 (1991); Alkema, et al., Nature 374:724–727 (1995)). A model for the function of chromosomal proteins leads one to conclude that they form multimeric complexes which determine the degree of condensation of the surrounding chromatin region depending on the balance between activators and repressors in the complex (Locke, et al., Genetics 120:181–198 (1988)). A shift in this equilibrium, caused by overexpression of one of the components of the complex, exhibited a new distribution of eu- and heterochromatic regions (Buck and Shore, Genes & Dev. 9:370–384 (1995); Reuter and Spierer, BioEssays 14:605–612 (1992); Eissenberg, et al., Genetics 131:345–352 (1992)) which can destabilize the chromatin structure at predetermined loci, and lead to a transition from the normal to the transformed state.
In spite of the characterization of HRX/ALL-1 and bmi as protooncogenes which are capable of changing the chromatin structure, knowledge of mammalian gene products which interact with chromatin is still very limited. By contrast, by genetic analyses of PEV in Drosophila, about 120 alleles for chromatin regulators have been described (Reuter and Spierer, BioEssays 14:605–612 (1992)).
Recently, a carboxy-terminal region was identified with similarity in the sequence to a positive (trx (trx-group)) and a negative (E(z) (Pc-group)) Drosophila chromatin regulator (Jones and Gelbart, MCB 13(10):6357–6366 (1993)). Moreover, this carboxy terminus is conserved in Su(var)3-9, a member of the Su(var) group, and a dominant suppressor of chromatin distribution in Drosophila (Tschiersch, et al., Embo J. 13(16):3822–3831 (1994)).
Genetic screens for suppressors of position effect variegation (PEV) in Drosophila and S. pombe have identified a subfamily of approximately 30–40 loci which are referred to as Su(var)-group genes. Interestingly, several histone deacetylases, protein phosphatase type 1 and S-adenosyl methionine synthetase have been classified as Su(var)s. In contrast, Su(var)2-5 (which is allelic to HP1), Su(var)3-7 and Su(var)3-9 encode heterochromatin-associated proteins. Su(var) gene function thus suggests a model in which modifications at the nucleosomal level may initiate the formation of defined chromosomal subdomains that are then stabilized and propagated by heterochromatic SU(VAR) proteins. Su(var)3-9 is dominant over most PEV modifier mutations, and mutants in the corresponding S. pombe clr4 gene disrupt heterochromatin association of other modifying factors and result in chromosome segregation defects. Recently, human (SUV39H1) and murine (Suv39h1 and Suv39h2) Su(var)3-9 homologues have been isolated. It has been shown that they encode heterochromatic proteins which associate with mammalian HP1. The SU(VAR)3-9 protein family combines two of the most evolutionarily conserved domains of ‘chromatin regulators’: the chromo and the SET domain. Whereas the 60 amino acid chromo domain represents an ancient histone-like fold that directs eu- or heterochromatic localizations, the molecular role of the 130 amino acid SET domain has remained enigmatic. Overexpression studies with human SUV39H1 mutants indicated a dominant interference with higher-order chromatin organization that, surprisingly, suggested a functional relationship between the SET domain and the distribution of phosphorylated (at serine 10) H3.
The experiments of the present invention show that mammalian SUV39H1 or Suv39h proteins are SET domain-dependent, H3-specific histone methyltransferases (HMTases) which selectively methylate lysine 9 of the H3 N-terminus. Methylation of lysine 9 negatively regulates phosphorylation of serine 10 and reveals a ‘histone code’ that appears intrinsically linked to the organization of higher-order chromatin.